Efﬁcacy of Azadirachta indica and Punica granatum Extracts in the Control of Cuminum cyminum Wilt Disease Caused by Fusarium oxysporum f. sp. cumini

: Punica granatum and Azadirachta indica are plants rich in phytochemicals, which directly contribute to antioxidant activity. The aim of this study was to test A. indica and P. granatum against Fusarium oxysporum f. sp. cumini (Foc), the causal pathogen of Fusarium wilt in cumin plants, in vivo and in vitro. After screening different concentrations of both plants, three concentrations (250, 500, and 1000 µ g · mL − 1 ) of P. granatum and A. indica were selected to study their effectiveness against Fusarium wilt in cumin plants. The in vitro study showed that both extracts have the ability to reduce mycelium growth of the pathogen with different degrees of efﬁcacy, but less than the positive control. Under greenhouse conditions, all treatments of cumin plants signiﬁcantly reduced Fusarium wilt compared to the infected control. The most effective concentration for P. granatum was 1000 µ g · mL − 1 . The use of both extracts signiﬁcantly increased the fresh and dry weight of cumin plants (g plant − 1 ) compared to infected plants. Total phenols and ﬂavonoids increased in inoculated cumin plants after treatment with both extracts. The results revealed that both extracts are rich in phytochemicals and possess potent in vitro antioxidant activity. Both are rich in carbohydrates, saponins, amino acids, proteins, alkaloids, and terpenoids. In conclusion, the application of methanolic extracts of P. granatum and A. indica can provide an alternative to chemical fungicides to mitigate the Fusarium wilt of cumin and, therefore, future studies should focus on the study of both extracts on different pathogens, as well their ability to reduce disease under ﬁeld conditions.


Introduction
Many vegetables, field crops, flowers, and plantation crops suffer severe losses due to Fusarium wilt [1]. According to previous studies, losses from cumin wilt have been estimated to range from 5 to 60% [1]. Warm winter climes are known to favor Fusarium wilt, especially when conditions are dry. Fusarium wilt disease is one of the most important cumin diseases [2]. The pathogen is seed-and soil-borne, and it is widely prevalent in almost all cumin-growing countries [1]. Chemical fungicides, Vitavax (carboxin)-thiuram or Vitavax-captan, are methods that provide a major deterrence to Fusarium wilt [3], but they are expensive and are known to be hazardous to human health and the environment [4]. Therefore, there is a pressing urgency for less expensive, more environmentally friendly methods of controlling Fusarium wilt.
Certain higher plant products, most notably, phytochemicals, can be a source of antimicrobial resistance, and have attracted attention due to their biodegradability and safety for human health, among other attractive properties [5]. According to Sharma and Bohra [6], Boerhavia diffusa leaf extract effectively inhibited the growth of Fusarium

Cumin Seeds and Growth of Plants
The cumin seeds (Cuminum cyminum L. cultivar Balady) used in this study were obtained from the Department of Ornamental Plants, Faculty of Agriculture, Egypt. Ten seeds were sown in each plastic pot filled with a pasteurized mixture of soil and sand (4:1 w/w), measuring 30 cm in diameter (2.4 kg). The seedlings were kept in the greenhouse at 50-70% relative humidity (RH) and temperatures between 25 and 30 • C, and they received about 12 h of natural light and 12 h of darkness daily. Each pot of plants was given roughly 50 mL of NPK (12:4:6) fertilizer suspension (1 g L −1 ) each week, in addition to the necessary amounts of water.

Source of Pathogen
A highly virulent strain was identified as Fusarium oxysporum f. sp. cumini (FO7) and deposited in the GenBank sequence database under accession number OM918708.1. This isolate was isolated previously by the authors. The isolate was grown on potato dextrose agar (PDA) obtained from HiMedia company, India, on plates (15 mL/plate) incubated at 27 • C for 7 days.

Preparation of Plant Extracts
Two plant species, Azadirachta indica and Punica granatum, were collected from different places from the Assiut Governorate, Egypt. The Azadirachta indica leaves and the fruit peel of Punica granatum were used. Plant materials were dried at room temperature for 15 days, then ground into fine powders in a grinder and used for the preparation of the methanolic extracts by maceration in 80:20 methanol:water at a 1:10 w/v ratio of sample to solvent, and kept under continuous shaking for three days at room temperature. Whatman No. 1 filter paper was used to filter the macerated materials after they had been filtered through two layers of cheesecloth. Using a rotary evaporator (Heidolph VV2000), the filtrates were mixed, concentrated, and then freeze-dried with a Telstar-LyoQuest plus-55 lyophilizer. The extract's yield was calculated and kept in opaque glass tubes at −20 • C [19,20].

In Vitro Antifungal Activity
Different volumes of crude methanol extracts were added to the PDA media before being poured into Petri plates in order to achieve extract concentrations of 250, 500, and 1000 µg·mL −1 . The plates were then inoculated with 2 mm fungal plugs in the center and cultivated for 10 days at 28 • C [21]. Calculations were used to determine the pathogen's percentage of inhibited radial growth. Five plates per replication were used to replicate each treatment three times. The commercial fungicide hymexazol [3-hydroxy-5-methylisoxazole 5-methylisoxazol-3(2H)-one hymexazole] was used as the positive control (1000 µg·mL −1 ). Methanol at 8% was used as the negative control. The percentage of reduction (Mr) of the colony diameter by each extract was computed following the technique used by Nduagu et al. [22]: where Mr = % reduction in colony diameter, M1 = colony diameter in the untreated medium (negative control), and M2 = colony diameter in the treated medium.

Greenhouse Conditions
The fungal inoculum of F. oxysporum f. sp. cumini (Foc) was prepared as follows: The Foc isolate was grown on a sterilized grain sorghum medium (200 mL + 150 g + 4 g + 4 g + 50 g of water, grain sorghum, sucrose, and clean sand, respectively) and incubated for two weeks at 27 • C [23]. The inoculum was mixed with the soil at 3% (w/w), and then irrigated three times over one week before transplanting to ensure an even distribution and growth of the pathogen isolate. Seven days before planting, sterilized sandy clay soil that had been inoculated with 3% (w/w) fungal inocula was placed in plastic pots (30 cm in diameter) [16]. Disinfected cumin seeds were soaked in the solutions of each peel methanol extract of P. granatum (Pme) and leaf methanol extract of A. indica (Lme) for 12 h before being seeded at a rate of 10 seeds per infested pot. Cumin seeds were also soaked in 8% methanol for the same amount of time and sown at the same rate in pathogen-infested soil as part of the control treatment. Each treatment consisted of five pots used as replicates. After 50 days from planting, the severity of the wilt was noted according to the method described by Ghoneem et al. [24]. Wilt severity was recorded 40 days after sowing. The disease index (%) = {[Σ (number of plants for each disease severity × disease rank)]/(total number of plants × the highest level)} × 100. The severity of Fusarium wilt in cumin was classified into five grades (0-5) according to the criteria of disease grades [24].

Phytochemical Screening Test
This was determined according to Reddy et al. [25].

Test for Phlobatannins
A total of 1 mL of 1% HCl was added to 1 mL of the extract before it was heated for 10 min. Phlobatannins were present when a precipitate of a red color formed.

Test for Carbohydrates
A total of 1 mL of the extract was mixed with three to five drops of Molisch reagent, and then 1 mL of concentrated sulfuric acid was carefully introduced via the test tube. After standing for two minutes, the liquid was diluted with 5 mL of pure water. Carbohydrates were detected by the formation of a red or dim violet ring at the liquid-liquid interface.

Test for Flavonoids
A total of 1 mL of the extract was added to a test tube, along with a few drops of 1% liquid ammonia, which caused the extract to become yellow, showing the presence of flavonoids.

Test for Alkaloids
A total of 2 mL of the sample and 2 mL of HCl were mixed. Following the addition of six drops of HCN and an additional two drops of picric acid, a pale-yellow cream color suggested the presence of alkaloids.

Test for Terpenoids
A total of 3 mL of conc. H 2 SO 4 and 2 mL of the sample were added, along with 2 mL of chloroform. The resulting red color showed the presence of terpenoids.

Test for Proteins
A small quantity of extract was added after 1 mL of ninhydrin had been dissolved in 1 mL of acetone. Protein was detected by the development of a purple color.

Detection of Saponins
Test for foam: a portion of the extract was rapidly shaken with water and any persisting foam was noted.

Test for Steroids
A total of 1 mL of concentrated sulfuric acid and 1 mL of chloroform were combined with 1 mL of extract. Ten drops of acetic anhydride were also added. The development of dark-red or dark-pink pigments was a sign that steroids were present.

Non-Enzymatic Assays
For the sample preparation, 1 gram of the root sample of each treatment was collected 35 days after the sowing date and washed with tap water extracted in 10 mL methanol:water (8:2 v/v). The suspension was shaken at 120 rpm for 2 h at 30 • C, then centrifuged at 1013 g for 5 min. The supernatant was collected and used for the determination of the total contents of phenols and flavonoids.

Total Phenol Content (TPC)
The level of TPC was determined in the prepared methanolic extract in accordance with the method reported by Malik and Singh [26]. All chemicals and reagents used in this experiment were purchased from Sigma-Aldrich (Waltham, MA, USA). The reaction mixture contained 50 µL of the sample methanolic extract mixed with distilled H 2 O (850 µL) and Folin-Ciocalteu reagent (100 µL). An aliquot of 500 µL of sodium carbonate (Na 2 CO 3 , 20%) was added to the mixture and left to react for 30 min before measuring the absorbance at 750 nm. A calibration curve of gallic acid was prepared using different concentrations, following the same procedure as for the sample analysis. TPC was expressed as mg gallic acid equivalent/g fresh weight of cumin samples. All treatments were repeated three times. TFC was evaluated using a modified method reported by Zhishen et al. [27]. An aliquot of 250 µL of the methanol extract was combined with 75 µL of 5% sodium nitrate (NaNO 2 ) solution and 1.25 µL of sterile distilled water in a 75 µL flask. After 6 min, 150 µL of 10% aluminum chloride (AlCl 3 ), 0.5 µL of 1 M sodium hydroxide (NaOH), and 275 µL of sterile distilled water were added to the solution. A calibration curve was created using known catechin concentrations analyzed the same way as the samples. The absorbance was recorded at 510 nm for the samples and the standard catechin, and the TFC was calculated and displayed as mg catechin equivalent/g fresh weight of orange samples.

Statistical Analysis
All tests were set up in a completely randomized design, and each experiment consisted of three replicates repeated twice. Using the statistical analysis method, data were subjected to an ANOVA [28]. The LSD test was used to compare the means at p < 0.05 levels.

Results and Discussion
Currently, the main method for disease control is through the use of fungicides [29], but chemical control is harmful to the environment and human health, and the control effects decrease gradually due to drug resistance in the pathogens. Therefore, it is necessary to search for natural and safer alternatives to chemicals. Many studies have recommended plant extracts to control fungal, bacterial, and helminthic diseases [16,30].

In Vitro Antifungal Activity
In this study, the antifungal activity of methanol extracts of two plant extracts, Azadirachta indica and Punica granatum, was tested. All concentrations of both plant extracts were effective in reducing the mycelial growth of Foc (Table 1). In general, P. granatum (Pme) exhibited the highest reduction in mycelium growth of the pathogen at 1000 µg·mL −1 , followed by 1000 for A. indica (Lme). The lowest reduction was achieved by A. indica at 250 µg·mL −1 . Tehranifar et al. [31] reported that the phenolic content of pomegranate peel extract was 2.8-fold higher compared to the leaf extract, and the high concentrations probably explained the antifungal activity of the extract. They found a number of biologically active compounds, such as flavonoids (quercetin, rutin, luteolin, pelargonidin, prodelphinidin, apigenin, etc.) and tannins (gallic acid, ellagic acid, punicalagin, and ellagitannin) [32]. Along the same lines, various studies have reported that plant extracts affected the growth of phytopathogenic fungi, as well as reduced disease severity, which may have been due to the secondary metabolites (phenolics, flavonoids, terpenoids, and alkaloids) [33][34][35].
Previous studies [36][37][38] mentioned that peel extracts of P. granatum were effective against many bacteria streptococci strains. Antibacterial activity may be due to the presence of the hydrolysable tannins and polyphenolics in the pomegranate extract, specifically, punicalagin and gallic acid [39,40]. This phenolic compound also has directly antifungal activity against Phytophthora spp. in vitro [41].
Azadirachtin is the main constituent of neem-based products which helps in the protection of crop plants from several pests and diseases [42]. Neem extracts are used to kill soil-borne pathogens, as well as seed-borne pathogens when used for seed treatment [42]. Plant extracts contain a mixture of many components, and their antifungal activity is probably not attributable to a single mechanism.
Values in the same column followed by different letters indicate significant differences among treatments according to the LSD test (p = 0.05).

Effect of Methanol Extract of Azadirachta indica and Peel Extract of Punica granatum on Disease Reduction under Greenhouse Conditions
The results, presented in Figure 1, show that the tested methanol extract of both plants at different concentrations significantly reduced the disease severity of Fusarium wilt disease in cumin plants under greenhouse conditions. The highest reduction was achieved by P. granatum at a concentration of 1000 µg·mL −1 , which showed nearly the same reduction in disease of 75% as hymexazole and A. indica at 1000 µg·mL −1 . The other concentrations of both extracts also could reduce the disease severity, but with the highest concentration (1000 µg·mL −1 ). Pme and Lme reduced the disease severity of Fusarium wilt of cumin ( Figure 1). Our findings were in general agreement with those of Hassenein et al. [43], who found that neem leaf extract sprays on tomato plants reduced the severity of Fusarium wilt. In the present study, both extracts reduced the Fusarium wilt of cumin when compared with the untreated control, and the same effect as fungicides was observed in the reduction of disease severity.

Vegetative Growth
The fresh weight (FW) and dry weight (DW) of the cumin plants increased significantly with both extracts at different concentrations compared to the control group (Figure 2). The height of the cumin plants increased significantly in all treatment groups compared to the control group (Figure 2). The highest increase was found with P. gran-

Vegetative Growth
The fresh weight (FW) and dry weight (DW) of the cumin plants increased significantly with both extracts at different concentrations compared to the control group ( Figure 2). The height of the cumin plants increased significantly in all treatment groups compared to the control group (Figure 2). The highest increase was found with P. granatum and A. indica treatments at 1000 µg·mL −1 , followed by 500 and 250 µg·mL −1 of P. granatum (98.8 and 96.7%, respectively). The same trend was observed for fresh and dry weight, which increased by 88 and 85.6%; this maybe correlated with the reduction in the disease severity. Many studies have noted that plant extracts increase the seed germination, as well as the vigor index, of ginger, garlic, and neem [44][45][46]. Additionally, Abo-Elyousr et al. [4], in their work on Calotropis procera, reported that tomato plants treated with an aqueous extract of C. procera showed increased fresh and dry weights of the tomato seedlings.

Effect of Different Concentrations of Azadirachta indica and Punica granatum on Phenol and Flavonoid Contents
From the data illustrated in Figure 3, the treatment of cumin plants with Pme and Lme at 1000 µg·mL −1 gave the highest flavonoid and phenol contents in inoculated or un-inoculated plants, followed by 500 µg·mL −1 and the lowest concentration of 250 µg·mL −1 , relative to the infected control plants.

Effect of Different Concentrations of Azadirachta indica and Punica granatum on Phenol and Flavonoid Contents
From the data illustrated in Figure 3, the treatment of cumin plants with Pme and Lme at 1000 µg·mL −1 gave the highest flavonoid and phenol contents in inoculated or uninoculated plants, followed by 500 µg·mL −1 and the lowest concentration of 250 µg·mL −1 , relative to the infected control plants.
Obtained results revealed that A. indica and P. granatum (250, 500, and 1000 µg·mL −1 ) increased the phenol content. In agreement with this, similar results have been reported in the literature. The authors of [4] found that the application of a plant extract (Calotropis procera) to tomato plants significantly reduced wilt severity compared to untreated controls. In this context, phytosterols, a group of steroidal alcohols, and phytochemicals naturally present in plants, have been found to display antifungal activity against Aspergillus, Penicillium, and Fusarium [47]. Phenolic compounds are toxic to phytopathogenic fungi, and the accumulation of these bioactive ingredients at infection sites has been associated with pathogen development restriction [48].

Effect of Different Concentrations of Azadirachta indica and Punica granatum on Phen Flavonoid Contents
From the data illustrated in Figure 3, the treatment of cumin plants with Pm Lme at 1000 µg·mL −1 gave the highest flavonoid and phenol contents in inoculat un-inoculated plants, followed by 500 µg·mL −1 and the lowest concentration o µg·mL −1 , relative to the infected control plants.

Phytochemical Screening Test
The results of the qualitative phytochemical analysis ( Table 2) of P. granatum peel and Azadirachta indica extracts showed that Pme and Lme are rich in carbohydrates, saponins, amino acids, proteins, alkaloids, and terpenoids. P. granatum peel extract is rich in carbohydrates, amino acids, proteins, alkaloids, and terpenoids. Phytochemical screening results showed that the methanol peel and leaf extract of both plants are rich in amino acids, flavonoids, alkaloids, and terpenoids, and fairly rich in saponins and flavonoids. Peel extract is rich in carbohydrates and saponins, and fairly rich in flavonoids. The secondary metabolites contribute significantly towards the biological activities, such as antioxidant and antidiabetic activities. Phytochemicals are mostly rich in green leaves, where they prevent free-radical formation. Hence, the presence of phytochemicals in the extracts might contribute to their beneficial activities [49]. Methanol extracts of P. granatum exhibited in vitro antioxidant activity in a concentrationdependent manner. Ascorbic acid was used as the standard chemical for comparing the antioxidant activity. Dietary antioxidants such as ascorbate, carotenoids, and tocopherol from natural compounds could reduce oxidative stress. Medicinal plants are a natural source for antimicrobial substances, which have fewer side effects and an ability to scavenge free radicals.

Conclusions
The seeds of cumin plants treated with the methanol extract of Azadirachta indica and Punica granatum at different concentrations reduced F. oxysporum f. sp. cumin radial growth in vitro; thus, the cumin seed pretreatment reduces the disease severity and increases the vegetative growth of cumin seedlings under greenhouse conditions. Seeds treated with both extracts had increased flavonoid and phenol contents in cumin roots at 35 days after the sowing date. Our results show that Azadirachta indica and Punica granatum at different concentrations are promising ecofriendly substances for use against F. oxysporum under greenhouse conditions.